Dot blot/Western background signal problem - (May/14/2015 )
I have been performing a dot blot where I probe for my protein of interest, then strip and reprobe for the loading control (I haven't been blocking after stripping, just incubating in primary and secondary in blocking buffer). My problem I that I get very high background signal to begin with and then after stripping and reprobing I get exactly the same background signal for the loading control! How would this happen if am incubating again in antibodies in blocking buffer? Is it just that the stripping isn't working very well?
proper stripping should just strip the antibodies from the blot.
if you are seeing high background when using different primaries then you probably have the secondary antibody binding to the blocking agent (eg if you use milk to block with an avidin-biotin secondary).